The liver has an essential role in protein metabolism; the degradation of amino acids occurs almost exclusively in the liver. Degradation is a process by which excess amino acids are catabolized and then used for energy or stored as fat. Degradation begins in the liver with deamination of the amino acids. The ammonia formed by deamination is removed from the blood and converted by the liver into urea, which is then excreted by the kidneys and intestines. In severe liver disease or damage, the ammonia that is normally converted to urea and excreted by the liver is allowed to accumulate to toxic levels in the blood.
Hepatocytes are parenchymal liver cells, which are the chief cells of the functional unit of the liver, the liver lobule. In the liver lobule, these cells are arranged hub-like around a central vein, with one side of the polyhedral hepatocyte facing the hepatic sinusoids, the capillary system of the liver, and the other side facing the bile canaliculi. Incoming portal and arterial blood enters the sinusoids, then passes through the liver lobule, where many substances are exchanged between the hepatocytes and the blood. The blood then courses on to the central vein, with special substances filtering into the bile ductules. Endothelial and Kupffer (Littoral) cells form the walls of the sinusoids.
There is considerable evidence that oxidative stress is a contributing factor in the pathogenesis of a variety of liver diseases. Evidence developed over the last several years has suggested that acute liver injury may frequently be caused by free-radical formation, and that these toxic radicals may injure the cell membranes of hepatocytes through lipid peroxidation or by other means. Lipid peroxidation can affect the composition and function of membranes, and ultimately every aspect of cell metabolism. Lipid peroxidation weakens the integrity of the cell membrane, which ultimately kills the cell.
Polyunsaturated fatty acids are particularly sensitive to oxidative stress and are easily damaged in the presence of oxidants such as partially reduced oxygen species (oxygen radicals). Possible sources of the toxic radical species may involve the activity of several liver enzyme systems, including, for example, acetaldehyde oxidase, xanthine dehydrogenase/oxidase, cytochrome p-450IIE1, and oxidases from infiltrating inflammatory cells.
It is believed that free radicals play a role in triggering the fibrosis and cirrhosis cascade. For example, the mechanism of liver damage by carbon tetrachloride is believed to be free radical mediated. The carbon tetrachloride molecule is dehalogenated by cytochrome P-450 to a trichloromethyl free radical. This trichloromethyl free radical adds molecular oxygen to form the trichloromethyl peroxyl radical. This reactive compound removes hydrogen atoms from unsaturated lipids, creating carbon-centered lipid radicals. These lipid radicals add molecular oxygen to produce lipid peroxyl radicals, which induces the process of lipid peroxidation. The membrane damage caused by lipid peroxidation, if not halted, results in the death of the cell due to severe damage to the cell membrane. The cell's natural defenses include scavengers such as vitamin E (.alpha.-tocopherol), dihydrolipoic acid, and superoxide dismutase. When these scavengers are exhausted, the damage caused by lipid peroxidation is unchecked.
The final stage of many types of liver injury is cirrhosis. Cirrhosis is a chronic liver disease wherein liver function is impaired due to extensive scar tissue. Cirrhosis occurs when the regeneration of new hepatocytes, bile ductules, vascular channels, and reticulin substance alters the normal flow of blood, bile, and hepatic metabolites. Any chemical or organism that causes liver destruction and irregular patchy regeneration will predispose to cirrhosis.
Oxidative stress, in association with ischemia and subsequent reperfusion that occurs with the harvesting and preservation of livers for transplantation, is also a major factor in primary liver graft malfunction. The harvesting of normal donor livers in viable condition is a crucial component of the transplantation process. Livers are considered viable for transplantation after only a limited time ex vivo, despite the introduction of solutions that may help to preserve them. Livers that remain untransplanted for 12 or more hours have a much higher incidence of graft failure secondary to oxidative stress. Even livers that are transplanted after a relatively short period of time following harvesting are subject to oxidative stress.
Liposomes are composed of various phospholipids surrounding an aqueous space which space is usually impermeable to the outside environment. Most liposome formulations have a strong affinity to the liver, and appear to be removed from the liver by the Kupffer cells. Liu and Huang, "Size Homogeneity of a Liposome Preparation is Crucial for Liposome Biodistribution in Vivo," J Liposome Res. 2: 57-66, 1992. This affinity is lipid dependent. By varying the size and lipid composition of the liposome, it is possible to alter the affinity to the various populations of cells within the liver itself. Scherphaf, et al., "Targeting of liposomes to liver cells". Drug Carrier Systems. Edited by F.H.D. Roerdink and A.M. Koon, 1989 John Wiley & Sons, Ltd., 281-291. Other methods for targeting liposomes to a particular target organ, such as the liver, utilizing antibodies, galactocerebrosides, and/or lipid composition, are known to those skilled in the art. See Roerdink, et al., "The Involvement of Parenchymal, Kupffer, and Endothelial Liver Cells in the Hepatic Uptake of Intravenously Injected Liposomes," Biochemica et Biophysica Acta, 1981, 677: 79-89; Spanjer, et al., "Intrahepatic distribution of small unilamellar liposomes as a function of liposomal lipid composition," Biochemica et Biophysica Acta, 1986, 863: 224-230.
Currently available treatments for liver disorders are not able to effectively concentrate a therapeutic agent in the target organ. Generally, when a pharmacologically active material is provided to a host, the material is distributed fairly evenly throughout the body, thus diluting its effect at the target organ. To achieve a reasonable effect, a large dose of drug must be administered. Large dosages are a problem when the drug is toxic to other organs. For example, chemotherapeutic agents designed to treat cancer or fulminant infections are limited by the hepatic toxicity of the therapeutic agent.
The present invention overcomes limitations in the prior art by utilizing liposome carrier systems, methods and pharmaceutical compositions that target the liver preferentially with high concentrations of at least one therapeutic agent.